There are over two hundred sequenced members of the superfamily of proteins called P450. They have been identified in bacteria, fungi, plants, and animals. In mammals, these enzymes are extremely important in drug detoxification, steroidogenesis, and carcinogenesis, while in some bacteria they participate in the assimilation of organic compounds. P450 monooxygenases can be divided into two classes depending the required redox partner: class I P450s requiring an iron sulfur protein and a reductase are found in bacteria and in the mitochondria of eukaryotes, and class II P450s requiring only an FAD/FMN-containing reductase are found predominantly as membrane-bound, microsomal proteins in eukaryotes. Until recently the only known x-ray crystal structure for a P450 was that of P450cam, a soluble class I P450 which hydroxylates camphor. Now, this research group has determined the structure of two more P450s: another soluble bacterial class I P450, P450terp, and most importantly, a soluble bacterial class II P450, P450BM-P. P450BM-P is the P450 domain of the naturally occurring fusion protein P450BM-3 which contains both a P450 domain and a reductase domain. P450cam, P450terp, and P450BM-3 are members of different gene families and have three different physiological substrates. The three dimensional structures of these proteins, while generally similar, are different, especially in regions involved in substrate- and redox-partner binding. Thus, while P450cam and P450terp serve as good models for class I P450s, P450BM-3 is the only known model for a class II P450, and as such, is important in understanding the mechanism of and protein structure required for electron transfer in the eukaryotic microsomal P450s. Additionally, since the substrates of all three are different, with P450cam and P450terp being very specific, and P450BM-P being a fatty acid monooxygenase which will oxidize a variety of other compounds, the structural features which control the specificity of oxidation can be investigated. Finally, substrate access to the active site in P450cam is not apparent from its three-dimensional structure; however, the access channel is open in the structure of P450BM-P and amenable to the study of substrate recognition and access to the active site. To these ends, this research project will be focused on the following Specific Aims: A. Can the stereo- and regiospecificity of the oxidation reactions catalyzed by P450BM-3 be altered by rational redesign of the active site? B. What role does the hydrophobic substrate recognition region, at the mouth of the substrate access channel, play in controlling the substrate specificity of P450BM-P? C. Does the movement of and the interaction between the beta-sheet rich and the alpha-helical rich domains of P450BM- P control access of substrates into the substrate access channel, and hence, to the active site? D. Is the interaction of the P450 with its physiologically relevant redox partner controlled by the "eukaryotic" insertion sequences, which include the J' helix and the two 3/10 helices following the K' helix; and is a change of the putative redox-partner docking region able to alter the specificity for the redox partner? E. Can P450BM-P be converted from a soluble protein to a membrane-bound form and how will this effect its substrate binding and its interaction with redox partners?